Background: The mechanisms by which viruses cause asthma exacerbations are not precisely known. Previously, we showed that, in ovalbumin (OVA)-sensitized and -challenged mice with allergic airway inflammation, rhinovirus (RV) infection increases type 2 cytokine production from alternatively-activated (M2) airway macrophages, enhancing eosinophilic inflammation and airways hyperresponsiveness. In this paper, we tested the hypothesis that IL-4 signaling determines the state of macrophage activation and pattern of RV-induced exacerbation in mice with allergic airways disease.

Figure 2: Airway inflammation and airway hyperresponsiveness in OVA-treated wild-type and IL-4R KO mice. Eight-week old wild-type or IL-4R KO mice were treated with PBS or OVA and inoculated with sham or RV. Bronchoalveolar lavage was performed 24 hour post-inoculation. After counting the total number of cells, cytospins were performed and stained with hematoxylin and eosin, and differential counts determined from 200 cells. The identity of neutrophils and eosinophils was confirmed by immunofluorescence staining for neutrophil elastase and major basic protein (not shown). (A) RV infection increases the total number of BAL cells per lung in OVA-sensitized and -challenged wild-type and IL-4R KO mice. (B) RV infection increases the number of airway neutrophils per lung in OVA-treated wild-type and IL-4R KO mice. The neutrophil response was significantly higher in IL-4R KO mice. (C) RV infection increases the number of airway eosinophils per lung in OVA-treated wild-type mice. The eosinophil response was significantly attenuated in IL-4R KO mice. (Mean ± SEM, n = 3, *different from medium, p < 0.05, one-way ANOVA; †different from wild-type, p < 0.05, one-way ANOVA.) (D & E) Airway cholinergic responsiveness was assessed by measuring changes in total respiratory system resistance in response to increasing doses of nebulized methacholine. Data from wild type (D) and IL-4R KO mice (E) are shown. (Mean ± SEM, n = 4-6 in each group, *different from sham, P < 0.05, two-way ANOVA; †different from PBS, P < 0.05, two-way ANOVA).

Mentions:
Next, we examined the effect of IL-4R KO in mice sensitized and challenged with OVA and infected with RV. As previously [17], RV alone had modest effects on airway neutrophilic inflammation and responsiveness (not shown). As expected, OVA treatment of wild-type mice significantly increased the total number of BAL cells (Figure 2A), augmenting the number of neutrophils and eosinophils (Figure 2B and C). RV infection of wild-type mice further increased lung inflammation, resulting in an additional 2-fold increase in BAL cells. Both neutrophils and eosinophils were increased in the BAL following RV infection. In IL-4R KO mice, OVA sensitization and challenge was also accompanied by a significant increase in BAL inflammatory cells (Figure 2A). The inflammatory cells consisted nearly exclusively of neutrophils, and the number of eosinophils in the airways was significantly reduced compared to wild-type mice (Figure 2B and C). When OVA-treated IL-4R KO mice were infected with RV, neutrophil infiltration further increased (Figure 2B). In contrast to wild-type mice, RV did not induce eosinophilic inflammation. Finally, we found that, similar to wild type mice, IL-4R KO mice showed OVA-induced airway hyperreactivity which was further exacerbated by RV (Figure 2D and E). (There was no difference in airway reactivity between the wild-type and IL-4R KO OVA/RV groups.) Together, these results show that IL-4 receptor signaling is not required for allergen-induced airway inflammation or hyperresponsiveness. Instead, the immune responses to OVA challenge and RV infection were differentially regulated in the absence of IL-4R signaling, accentuating neutrophilic rather than eosinophilic inflammation.

Figure 2: Airway inflammation and airway hyperresponsiveness in OVA-treated wild-type and IL-4R KO mice. Eight-week old wild-type or IL-4R KO mice were treated with PBS or OVA and inoculated with sham or RV. Bronchoalveolar lavage was performed 24 hour post-inoculation. After counting the total number of cells, cytospins were performed and stained with hematoxylin and eosin, and differential counts determined from 200 cells. The identity of neutrophils and eosinophils was confirmed by immunofluorescence staining for neutrophil elastase and major basic protein (not shown). (A) RV infection increases the total number of BAL cells per lung in OVA-sensitized and -challenged wild-type and IL-4R KO mice. (B) RV infection increases the number of airway neutrophils per lung in OVA-treated wild-type and IL-4R KO mice. The neutrophil response was significantly higher in IL-4R KO mice. (C) RV infection increases the number of airway eosinophils per lung in OVA-treated wild-type mice. The eosinophil response was significantly attenuated in IL-4R KO mice. (Mean ± SEM, n = 3, *different from medium, p < 0.05, one-way ANOVA; †different from wild-type, p < 0.05, one-way ANOVA.) (D & E) Airway cholinergic responsiveness was assessed by measuring changes in total respiratory system resistance in response to increasing doses of nebulized methacholine. Data from wild type (D) and IL-4R KO mice (E) are shown. (Mean ± SEM, n = 4-6 in each group, *different from sham, P < 0.05, two-way ANOVA; †different from PBS, P < 0.05, two-way ANOVA).

Mentions:
Next, we examined the effect of IL-4R KO in mice sensitized and challenged with OVA and infected with RV. As previously [17], RV alone had modest effects on airway neutrophilic inflammation and responsiveness (not shown). As expected, OVA treatment of wild-type mice significantly increased the total number of BAL cells (Figure 2A), augmenting the number of neutrophils and eosinophils (Figure 2B and C). RV infection of wild-type mice further increased lung inflammation, resulting in an additional 2-fold increase in BAL cells. Both neutrophils and eosinophils were increased in the BAL following RV infection. In IL-4R KO mice, OVA sensitization and challenge was also accompanied by a significant increase in BAL inflammatory cells (Figure 2A). The inflammatory cells consisted nearly exclusively of neutrophils, and the number of eosinophils in the airways was significantly reduced compared to wild-type mice (Figure 2B and C). When OVA-treated IL-4R KO mice were infected with RV, neutrophil infiltration further increased (Figure 2B). In contrast to wild-type mice, RV did not induce eosinophilic inflammation. Finally, we found that, similar to wild type mice, IL-4R KO mice showed OVA-induced airway hyperreactivity which was further exacerbated by RV (Figure 2D and E). (There was no difference in airway reactivity between the wild-type and IL-4R KO OVA/RV groups.) Together, these results show that IL-4 receptor signaling is not required for allergen-induced airway inflammation or hyperresponsiveness. Instead, the immune responses to OVA challenge and RV infection were differentially regulated in the absence of IL-4R signaling, accentuating neutrophilic rather than eosinophilic inflammation.

Background: The mechanisms by which viruses cause asthma exacerbations are not precisely known. Previously, we showed that, in ovalbumin (OVA)-sensitized and -challenged mice with allergic airway inflammation, rhinovirus (RV) infection increases type 2 cytokine production from alternatively-activated (M2) airway macrophages, enhancing eosinophilic inflammation and airways hyperresponsiveness. In this paper, we tested the hypothesis that IL-4 signaling determines the state of macrophage activation and pattern of RV-induced exacerbation in mice with allergic airways disease.